The process of metastasis is one of the most destructive characteristics of cancer, yet it is still poorly understood. Formation of metastasis comprises several distinct steps: cancer cells first detach from the primary tumor and then enter the bloodstream where they circulate freely in the body and eventually adhere to vessel walls in distant organs where they can form secondary tumors. A recent study discovered that a co-treatment of two common drugs that interfere with cellular metabolism, metformin and 2-deoxy-D-glucose, induced cellular changes resembling those observed in metastasis: the drugs induced detachment of certain breast cancer cells and their proliferation in the floating state. In this study, we investigated if this treatment also induces other changes that are related to metastasis, i.e., if the detached cells are softer and if they are more prone to adhesion than control cells. The results of our in vitro experiments showed that this was indeed the case and thus indicate possible relations between metabolism and metastatic potential. While the results of this study cannot be directly projected to cancers in vivo, they present new observations that can be important for the analysis of cancer cell detachment and anchorage-independent growth.Metastatic cancer cells can overcome detachment-induced cell death and can proliferate in anchorage-independent conditions. A recent study revealed that a co-treatment with two drugs that interfere with cell metabolism, metformin and 2-deoxy-D-glucose, promotes detachment of viable MDA-MB-231 breast cancer cells. In the present study, we analyzed if these detached viable MDA-MB-231 cells also exhibit other features related to cancer metastatic potential, i.e., if they are softer and more prone to adhere to epithelial cells. The cell mechanics of attached cells and floating cells were analyzed by optical tweezers and cell deformability cytometry, respectively. The adhesion was assessed on a confluent monolayer of HUVEC cells, with MDA-MB-231 cells either in static conditions or in a microfluidic flow. Additionally, to test if adhesion was affected by the state of the epithelial glycocalyx, HUVEC cells were treated with neuraminidase and tunicamycin. It was found that the treated MDA-MB-231 cells were more prone to adhere to HUVEC cells and that they were softer than the control, both in the floating state and after re-seeding to a substrate. The changes in the HUVEC glycocalyx, however, did not increase the adhesion potential of MDA-MB-231. One of the most lethal features of various cancer types is the formation of metastasis, a process where cancer cells spread from the primary tumor to other organs or tissues to form secondary tumors. The metastatic process involves several distinct steps, starting with detachment from the primary tumor and entry into the bloodstream by intravasation ,2,3,4,5.Normal epithelial cells are anchorage dependent, and when they detach from the extracellular matrix, they enter a particular type of cell death called anoikis . HoweverIn the next step of the metastatic process, the adhesion of CTCs to endothelial cells, it is crucial that both cell types express adequate ligands and receptors . An impoAs shown by Bizjak et al. , co-treaHUVEC (ATCC) were grown in serum-reduced minimum essential medium (MEM)  supplemented with 5% fetal bovine serum (Life Technologies) and MDA-MB-231 cells (ATCC) in RPMI medium  supplemented with 10% fetal bovine serum (Life Technologies) and 4.5 g/L glucose. Antibiotics (streptomycin-penicillin) (Life Technologies) were added to both media. To obtain floating MDA-MB-231 cells, they were first grown in a standard T-25 cell culture flask. The control MDA-MB-231 cells were detached by trypsinization 48 h after seeding and then harvested from the solution. The floating met2DG-treated MDA-MB-231 cells were treated with 5mM metformin and 0.6 mM 2-deoxy-D-glucose after seeding for 48 h and then harvested from the solution .For the experiments, HUVEC cells were treated one day after seeding with neuraminidase (Sigma-Aldrich) at 1 U/mL for 24 h and with tunicamycin (Sigma-Aldrich) at 5 µg/mL for 48 h. The concentrations and duration of treatments used were chosen based on the literature data and our preliminary experiments.4 cells/mL. The cell viability was tested on control, neuraminidase and tunicamycin treated HUVEC cells. The cells were plated in 96-well microtiter plates (TPP) at a concentration of 3 × 10H-tetrazolium) test. To each well, 20 μL of MTS  was added to the cell culture medium. After 2 h, the absorbance at 490 nm was measured using a Bio-Tek microplate reader . The absorption corresponded to the amount of the soluble formazan product that was formed, which is directly proportional to the number of viable cells. The viability was calculated as the ratio between absorbance at 490 nm of the treated and control cells. The cell viability was determined using the MTS -5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-24 cells/mL) into a custom-built experimental chamber with an uncoated, plasma-treated glass bottom. One day after seeding, the cells were left untreated or were treated with neuraminidase or tunicamycin. The assay was performed 48 h after seeding for control and neuraminidase treatments and 72 h for tunicamycin treatment. Floating MDA-MB-231 cells were obtained by trypsinization (control), met2DG treatment or by harvesting of cells cultured on polyHEMA (Sigma-Aldrich) coated flasks for 48 h. All MDA-MB-231 cells were stained after detachment with 0.5 µM calcein acetoxymethyl ester  for 15 min in the incubator. Thereafter, 1 × 104 cells/mL of stained MDA-MB-231 cells were seeded on the confluent monolayer of HUVEC cells and incubated for 20 min. Then the cells in the experimental chambers were washed thoroughly, so all weak and unattached cells were washed away. The samples were monitored under Eclipse Ti inverted microscope  with epi-fluorescence, and 20 random fields of views were recorded. The adhered cells were then counted with the help of the ImageJ Cell Count Plugin [HUVEC cells were seeded (3 × 10t Plugin .4 cells/mL) into an uncoated flow chamber (ibidi µ-slide I0.2 Luer). As in the static adhesion assay, the cells were whether left untreated or treated with neuraminidase or tunicamycin. For the adhesion assay, floating MDA-MB-231 cells (105 cell/mL) stained with 0.5 µM calcein AM were flown over the monolayers of HUVEC cells for 15 min. The flow was kept constant using a neMESYS syringe pump  at 0.5 µL/sec. Thereafter the cells were rinsed with the growth buffer at 1 µL/sec for 10 min, so all weakly attached and unattached MDA-MB-231 cells were washed away. The samples were examined under the Eclipse Ti inverted microscope (Nikon) with epi-fluorescence. Nine fields of view from the central area of the µ-slide at 4 different distances  from the microchannel entrance were imaged. The cells in the respective fields of views were then counted in the same way as in 2.3.HUVEC cells were seeded  were measured 72 h after seeding. For the measurements of re-seeded cells, control and met2DG-treated floating MDA-MB-231 cells were seeded into the chamber at the concentration of 3 × 104 cells/mL. The cells were left to attach to the substrate for one hour before the measurements were conducted. For the measurements of HUVEC cells, they were seeded into the chamber at a low concentration (1.5 × 104 cells/mL) so that they did not form a confluent layer even after 24 or 48 h of treatment with neuraminidase or tunicamycin.The stiffness of cells was measured with optical tweezers in a custom-build PDMS experimental chamber adhered to an uncoated glass bottom. Adhered MDA-MB-231 cells were seeded into the chamber at the concentration of 1.5 × 10Cell stiffness measurements were performed on an Eclipse Ti inverted microscope (Nikon) equipped with laser tweezers , as described in Zemljic Jokhadar et al. . In brieThe experimental chamber with cells was mounted on the microscope, and silica beads with a diameter of 5.06 µm  were added into the sample, where they were trapped by optical tweezers. The beads were then positioned near the selected cell. The bead center was approximately 5 µm above the surface, which was achieved with the piezo microscope stage . By moving the piezo stage with a constant velocity of 1 µm/s, the cell was pushed into the bead. The bead position was monitored in real-time with a digital camera  at 50 fps and by using a custom-written Matlab program . The piezo stage was automatically retracted after the bead was pushed for 0.5 µm from the center of the optical trap. The force was calculated fromThe cell stiffness was determined as the slope of the linear part of the force–deformation curve . The ind6 cells/mL. The cell suspensions were loaded into 1 mL syringe, which was fixed to the Nemesys pump  and connected to the Flic30 microfluidic chip . The flow rate of the cell suspension was set to 0.25 µL/s, and the flow rate of the buffer was set to 0.75 µL/s [The method for deformability cytometry was adopted from Otto et al. . Detache.75 µL/s . Before A of the cells by counting pixels inside the automatically detected contours and the perimeter l as contour length. Following the definition from Otto et al. [We processed the acquired images with custom-written image recognition software . The automatic shape detection employed background subtraction, Gaussian blur, automatic Otsu thresholding and was filtered according to the expected size and solidity of the obtained shapes. We determined the size (projected cell surface area) o et al. , the def4 cells/mL. One day after seeding, cells in uncoated wells were left untreated or were treated with met2DG for 48 h. Forty-eight hours after seeding, the suspension from uncoated and polyHEMA coated wells and 72 h from wells with met2DG treated MDA-MB-231 cells was collected and centrifuged. The supernatant was stored at −80 °C and then used for ELISA analyses. For the detection of cytokine, we used immune-enzyme test Human TNF alpha Uncoated ELISA  following the manufacturer’s instructions.MDA-MB-231 cells were seeded in uncoated or polyHEMA coated wells of a 6-well plate (TPP) at 5 × 10HUVEC cells were grown and treated with neuraminidase or tunicamycin as in other experiments. After the treatments, we incubated the cells with 4 µg/mL of wheat germ agglutinin, Alexafluor 488 conjugate  in the growth media for 15 min. Then we gently washed the cells and examined the samples under the Nikon ECLIPSE TE2000-E microscope  in the confocal mode (Nikon C1). HUVEC cells were grown and treated with neuraminidase or tunicamycin as in other experiments. HUVEC cells were seeded in 24-well culture plates for 24 h and then treated for 24 h with neuraminidase or for 48 h with tunicamycin.After the treatments, we incubated the cells with 4 µg/mL of wheat germ agglutinin, Alexafluor 488 conjugate  in the growth media for 15 min. Cells were washed with physiological saline and harvested by trypsinization. After trypsinization, cells were resuspended in cold PBS and measured with Attune N × T flow cytometer (Thermo Fisher Scientific). Three independent biological repeats were performed.For detached cells, MDA-MB-231 cells were plated on 6-well plates at 160,000 cells per well for cells treated with 5 mM metformin + 0.6 mM 2DG for 48. For cells grown on poly-HEMA, MDA-MB-213 cells were seeded in complete RPMI medium with 1 g/L glucose on poly-HEMA coated 6-well plates at 215,000 per well for 72 h. After treatment, detached cells population were collected, and attached cells were detached with trypsin. All cell populations were spun down and resuspended in Seahorse XF RPMI 1640-based Seahorse XF Glycolytic Rate Assay medium . The Seahorse Cell Energy Phenotype Assay (Agilent) was performed following manufacturer instructions, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using Seahorse Analyser XFp .p-values. For comparison of mean values between the treated and the control cells, the Student’s t-test was used. In both cases, p-values < 0.05 deemed significant.For comparison of median values between the treated and the control cells, the Mann–Whitney–Wilcoxon test  was used to calculate the An important step in the metastatic cascade is the adhesion of CTCs to endothelial cells. We, therefore, analyzed if floating MDA-MB-231 cells that were detached by met2DG treatment are more prone to adhere to normal HUVEC monolayer than the control MDA-MB-231 cells that were detached by the standard trypsinization protocol. The test was conducted in static conditions, as well as in a microfluidic system that mimics the shear flow in the vasculature. In the static conditions, floating MDA-MB-231 cells were placed over a HUVEC monolayer in the experimental chamber for 20 min and then thoroughly washed. In the microfluidic system, floating MDA-MB-231 cells were flown over a HUVEC monolayer in a microfluidic system for 15 min. The results showed that met2DG-treated MDA-MB-231 cells adhere significantly more to the HUVEC cells then control MDA-MB-231 cells in both the static conditions a and undWe analyzed the effects of met2DG treatment on stiffness of MDA-MB-231 cells in three different conditions with two different techniques. To test if met2DG treatment alone can alter cellular stiffness, we used optical tweezers to measure the stiffness of the attached control and met2DG-treated MDA-MB-231 cells. Second, we collected floating cells obtained after 48 h of met2DG treatment and control cells detached by trypsinization and performed deformability cytometry, where deformations of individual floating cells were measured as they were pumped at high velocity through a constricted channel. To test if trypsinization has an effect on cell stiffness, we also included cells grown in flasks coated with polyHEMA. Third, we re-seeded the floating cells and probed them again with optical tweezers. Both techniques are single-cell-based, but probing with optical tweezers measures cell stiffness at small deformations, i.e., at the level of the cell membrane, while microfluidic cell deformability assay measures deformations of whole cells. In the first case, we did not measure any significant difference in cell stiffness between control and met2DG-treated adhered MDA-MB-231 cells a. On theAs metformin and 2DG are metabolic inhibitors, we determined the cell energy phenotype of the floating met2DG-treated cells using Seahorse Analyser XFp . MDA-MB-We observed that Met2DG-treated cells had significantly altered cell energy phenotype, with almost completely suppressed mitochondrial respiration a compareIn the second part of our study, we examined the role of epithelial GLX in the adhesion of MDA-MB-231 cells to HUVEC cells. HUVEC GLX was treated by tunicamycin, which blocks N-glycosylation, and neuraminidase, which degrades the sialic parts of GLX. As the adhesion can be affected by cell stiffness, we also measured the stiffness of treated HUVEC cells with optical tweezers.We first evaluated the effects of treatments on the amount of GLX on HUVEC cells using confocal microscopy and flow cytometry by staining the cells with fluorescent WGA, which is a nonspecific GLX stain. The representative images obtained by confocal microscopy are shown in To quantify these results, we also analyzed the WGA-stained HUVEC cells with flow cytometry. After the treatments, the cells were harvested by trypsinization and introduced into the flow cytometer. The measured mean fluorescence intensity is presented in In the next step, we analyzed if the impaired HUVEC GLX influences the adhesion of MDA-MB-231 cells. The measurements were again conducted in static conditions and under a microfluidic flow. Control MDA-MB-231 cells (detached by trypsinization) were compared to the MDA-MB-231 cells detached by met2DG treatment . For allFinally, we tested if the treatments of HUVEC cells affected their stiffness. Again we employed the measurements with optical tweezers. While tunicamycin-treated cells showed no marked changes, the neuraminidase-treated cells were significantly stiffer than the control .Cancer cells can detach from the primary tumor, bypass anoikis and attach to the matrix of distant organs in the body, where they can form secondary tumors ,4,13. ThMDA-MB-231 human breast cancer cell line is commonly used to model late-stage breast cancer and has metastatic properties . A studySince deregulation of anoikis is linked to metastatic ability, our goal was to investigate if this subpopulation of met2DG-treated, viable, floating MDA-MB-231 cells has mechanical characteristics that are related to increased metastatic potential; specifically, we analyzed their stiffness and ability to adhere to endothelial monolayer. Additionally, we analyzed the effects of two drugs that disrupt epithelial GLX on MDA-MB-231 adhesion. In the first part of the study, we compared the adhesion of normal and met2DG-treated MDA-MB-231 cells to normal endothelial HUVEC cells. Since the adhesion of CTC to vessel walls is most prominent in post-capillary venules, where only low shear stress is present ,26, we cSeveral studies had shown that cancerous cells exhibit different mechanical properties than normal cells of the same tissue origin. For example, highly invasive malignant MDA-MB-231 cells are softer than benign human mammary gland cell line MCF-10 or MCF-7, which is a non-invasive malignant breast cancer cell line ,29,30. TIn parallel, we observed significantly altered cell energy phenotype of the floating met2DG-treated compared to control  MDA-MB-231 cells, with almost completely blocked mitochondrial respiration . On the GLX covers the endothelium, and in normal conditions, it forms a selective barrier between the endothelium and its environment by preventing adverse ligands the access to adhesion receptors. Therefore, in the second part of our study, we focused on the role of the endothelial GLX in the adhesion of MDA-MB-231 cells. We treated the HUVEC cells with two substances that affect the GLX on different levels. The endothelial GLX is composed of different glycoproteins, soluble proteins and residual chains, among others such as sialic acid, which are attached to core proteins . Sialic Flow-cytometric analysis of HUVEC cells labeled with fluorescent WGA confirmed that both treatments reduced the amount of GLX . InteresTreatments of HUVEC cells with neuraminidase and tunicamycin did not result in an increase in the adhesion of MDA-MB-231 cells . In factWe found that MDA-MB-231 cells that detach in vitro due to a combined treatment of two cell metabolism-interfering drugs, metformin and 2DG, are more prone to adhere to endothelial HUVEC cells than control MDA-MB-231 cells. They are also softer (more deformable) than the control cells both in suspension and after they re-attach to the glass substrate. While these features could be related to a higher metastatic potential, further studies are needed to confirm this connection in vivo.


Pochonia chlamydosporia is a fungal parasite of nematode eggs. Studies have shown that some strains of Pochonia chlamydosporia can promote plant growth and induce plants’ systemic resistance to root-knot nematodes by colonizing in their roots. This study aimed to verify the effect of the PC-170 strain on tomato growth and systemic resistance. Split-root experiments were conducted to observe the systemic resistance induced by PC-170. To explore the defense pathway that was excited due to the colonization by PC-170, we tested the expression of marker genes for defense pathways, and used mutant lines to verify the role of plant defense pathways. Our results showed that PC-170 can colonize roots, and promotes growth. We found a role for jasmonic acid (JA) in modulating tomato colonization by PC-170. PC-170 can activate tomato defense responses to reduce susceptibility to infection by the root-knot nematode Meloidogyne incognita, and induced resistance to some pathogens in tomatoes. The marker genes of the defense pathway were significantly induced after PC-170 colonization. However, salicylic acid (SA)- and jasmonic acid (JA)-dependent defenses in roots were variable with the invasion of different pathogens. Defense pathways play different roles at different points in time. SA- and JA-dependent defense pathways were shown to cross-communicate. Different phytohormones have been involved in tomato plants’ responses against different pathogens. Our study confirmed that adaptive JA signaling is necessary to regulate PC-170 colonization and induce systemic resistance in tomatoes. Plants are constantly subjected to various attacks from phytopathogenic fungi, bacteria, and plant-parasitic nematodes. Plants are not entirely passive, because the plants themselves have a series of corresponding immune responses, and they benefit from endophytic fungi to modulate their interactions with pathogens. In recent decades, some thought-provoking plant innate immune models have been put forward, such as the ‘Zigzag model’ in 2006, which indicated that there was co-evolution between pathogens and plants . The ‘InPochonia chlamydosporia belongs to the Clavicipitaceae family [P. chlamydosporia has the ability to colonize in the roots of both monocotyledonous and dicotyledonous plants, including barley [Arabidopsis thaliana [P. chlamydosporia has also exhibited multiple functions in promoting plant growth [P. chlamydosporia in plant root systems promotes growth and increases yield [Arabidopsis by P. chlamydosporia, and its ability to induce beneficial traits [P. chlamydosporia to induce systemic resistance to Meloidogyne incognita in tomatoes, but other studies have indicated that this response is plant-species-dependent [P. chlamydosporia, a number of secreted proteins have been identified that enabled a comprehensive understanding of the endogenous mechanisms and adaptive evolution of P. chlamydosporia [P. chlamydosporia induces plant defenses [e family ,8, whiche family ,10,11. Te family ,12,13,14g barley , tomatoeg barley , potatoeg barley , and Arathaliana . Interest growth ,19. Preves yield ,21. Studependent . Recentldosporia . Investidefenses .Phytohormones play essential roles during plant growth and development, and also coordinate the modulation of plant defenses . Some stPR) genes. The non-expressor of PR-1 (NPR1) mutants are insensitive to SA, fail to develop SAR, and exhibit enhanced disease susceptibility. For this reason, the expression of PR genes is frequently used as a marker of SAR development [PR-1a) is a protein that is strongly induced during the onset of systemic acquired resistance (SAR) in tobacco [PR1b1  gene is also strongly activated locally in tissues undergoing the hypersensitive response. Phenylalanine ammonia lyase (PAL) catalyzes the deamination of phenylalanine to trans-cinnamic acid, and is the first rate-limiting enzyme in the phenylpropanoid metabolism pathway. The expression of the PAL gene marks the beginning of SA biosynthesis. Silencing or epigenetically suppressing PAL genes inhibits the salicylate production, preventing the development of systemic acquired resistance in response to infection by the tobacco mosaic virus [NPR1, which was identified in Arabidopsis through genetic screens for SAR-compromised mutants, is an important transducer of the SA signal in disease resistance, and is essential for SA-induced PR-1a gene expression. NPR1 was reported as being a positive regulator of SA-dependent, defense-related gene expression, but negatively regulates JA-responsive gene expression [NPR1 modulates crosstalk between salicylate- and jasmonate-dependent defense pathways. LoxD and OPR3 are JA biosynthesis genes. In the biosynthesis of jasmonic acid (JA), the lipoxygenases (LOX) are a group of enzymes responsible for converting both linoleic and linolenic acids into 9- and 13-hydroperoxides. OPRs belong to a family of flavoproteins. OPR3 of tomato is a peroxisomal enzyme. The role of OPR3 in JA biosynthesis has been shown by genetic evidence provided by the JA-deficient mutants opr3 and dde1, both affected in OPR3. All genes encoding enzymes of JA biosynthesis are JA-inducible [PI II) and multicystatin (MC) are the typical JA-regulated, wound-related genes.Around the time of hypersensitive response (HR) development, the levels of salicylic acid (SA) increase, along with the induction of various defense-associated genes, including the pathogenesis-related  was isolated from the root-knot nematode M. incognita [Therefore, further research on beneficial rhizosphere microorganisms (such as endophytes) is important. There have been studies on the interaction between ncognita . This reP. chlamydosporia used in this study was isolated from Meloidogyne incognita eggs. The strain was stored in the CGMCC  under the preservation number 8860 [The PC-170 strain of ber 8860 . The PC-ber 8860 , were cuSolanum lycopersicum) cultivars Lichun, Castlemart, and Moneymaker were used in this study. These tomato varieties are not resistant to nematodes or various other pathogens, and are classified as susceptible varieties. Additionally, the JA-impaired mutant def1 in the Castlemart background [NahG in the Moneymaker background [DEF1 gene acts after lipoxygenase conversion of linolenic acid to hydroperoxylinolenic acid, but before the conversion of 12-oxo-phytodienoic acid (PDA) to JA. The def1 mutant is deficient in the DEF1 gene; thus, the octadecanoid pathway in the def1 mutant is blocked downstream of 13(S)-hydroperoxylinolenic acid (HPLA) action, but upstream of 12-oxo-phytodienoic acid (PDA) action. NahG is a gene encoding salicylate hydroxylase that removes endogenous SA by converting it to catechol. The NahG mutant expressing the NahG gene, encoding salicylate hydroxylase, did not accumulate SA in response to intercellular infiltration with washing fluid. Tomato seeds were surface-sterilized with 2% sodium hypochlorite, thoroughly rinsed with sterile water, and then germinated for 10 d in soil at 25 °C. Then, tomato seedlings were transplanted into soil in different nutrition bowls in a greenhouse according to the experimental requirements. The soil used in all experiments was made of peat soil , perlite, and vermiculite mixed in a ratio of 2:1:1, and then sterilized at 121 °C for 60 min after mixing. The plants received water according to their normal growth needs.The tomato (ckground  and the ckground  were useFusarium oxysporum f.s p. lycopersici (Sacc.) Snyder and Hansen, TFW) was grown on PDA plates with rifampicin. Tomato bacterial wilt  Yabuuchi et al., TBW) was preserved in sterile distilled water. Cucumber mosaic virus (CMV) and tomato mosaic virus (ToMV) were stored in the laboratory, and were inoculated regularly on the leaves of tobacco (Nicotiana tabacum L.).Tomato fusarium wilt (Meloidogyne incognita was carried out in a greenhouse with water spinach (Ipomoea aquatica Forssk.) as the growth material. We used tweezers to manually pick out the nematode eggs from the roots of the water spinach, disinfected them with 0.5% sodium hypochlorite, washed them with sterile water, collected them on a 25 μm sieve on a 90 mm plate, and stored them at 25 ± 2 °C. The second stage juveniles (J2s) were collected from the filter with a pipette every day. The number of J2s collected for use was determined according to the needs of the experiment.The long-term propagation of 2 spores·mL−1, 5 × 106 spores·mL−1, and 5 × 1010 spores·mL−1. Each nutrient bowl was filled with 50 g of soil mixed with 20 mL of prepared spore solution before tomato transplantation.The tomato variety used in the experiment was Lichun. Inoculation methods included fungus cake inoculation and spore fluid inoculation. Fungus cake inoculation was performed by using a punch to remove a 1 cm cylindrical piece of fungus cake of the PC-170 strain growing on a PDA plate, which had been cultured in a 28 °C incubator for 1 week, and then evenly burying four of these 1 cm fungus cake pieces in the rhizosphere of each tomato plant. The spore fluid inoculation was conducted by using sterile water to rinse the PDA plates inoculated with the PC-170 strain. The plates were rinsed several times to collect conidia that were growing on the PDA plates after culturing in an incubator at 28 °C for 1 week. Three different concentrations of spore liquid were used for inoculation: 5 × 10−1) and plated in growth-restricting medium  300 mg·L−1, and 50 mg·L−1 each of streptomycin sulfate and chloramphenicol). Total root colonization by P. chlamydosporia was evaluated using quantitative PCR (q-PCR). DNA was extracted from non-disinfected roots to assess total colonization, and the root material was collected. Roots (1 g) were freeze-dried and then ground in liquid nitrogen. Total DNA was extracted using the Plant Genomic DNA Kit (TIANGEN BIOTECH (BEIJING) CO., Ltd., Beijing, China). The DNA was quantified using a NanoDrop 2000C spectrophotometer . In qPCR experiments, tub1 primers were used to amplify a partial region of the tubulin gene of PC-170. Reactions were performed in 20 μL with 50 ng of root DNA, 500 nM primers, and 1 × SYBR Green Master Mix (TIANGEN BIOTECH (BEIJING) CO., Ltd.). All samples were diluted in 0.01% DEPC-treated water. Reactions were performed in triplicate in a Thermal Cycling StepOnePlus using the following profile: 95 °C for 15 min, 40 cycles at 95 °C for 10 s, 60 °C for 30 s, and 72 °C for 60 s. For standard curve construction, serial dilutions were prepared from 100 ng to 10 pg of genomic DNA from P. chlamydosporia PC-170 in 50 ng of tomato root DNA. The cycle threshold (CT) values obtained per well containing total DNA (root DNA) were correlated with CT values in the standard curve to calculate the quantity of fungal DNA vs. total DNA.To detect endophytic colonization, the roots of three plants were randomly selected at different time periods after the inoculation with PC-170, and then the roots were rinsed and disinfected with 1% sodium hypochlorite for 1 min, washed three times in sterile distilled water, and blotted onto sterile paper. Roots were then cut into 1 cm fragments . Nematode inoculation was performed on the 10th day after PC-170 inoculation. In all experiments, each tomato seedling was uniformly inoculated with 1000 J2s in the soil.M. incognita J2s (M); and (4) inoculation with PC-170 first, and then with M. incognita J2s (PC+M). The tomato variety used in the experiment was Lichun. All plants were placed randomly, with 30 plants per treatment. Three tomato plants were randomly selected and their roots were washed for observation. The fresh weight of shoots (FWS), dry weight of shoots (DWS), fresh weight of roots (FWR), and dry weight of roots (DWR) of the plant were measured after 45 d. The severity of nematode infection of the plants was determined at the same time. Root systems were carefully washed with tap water. Nematode performance was analyzed by counting gall numbers on roots at each timepoint, by visual inspection. Fecundity was determined by counting the number of egg masses and the number of eggs per egg mass after 45 days. The number of egg masses was analyzed by visual inspection, and the percentage of galls showing egg masses was calculated. The number of eggs per egg mass was determined by selecting 10 egg masses randomly from each root system and soaking them in 1% bleach solution for 5 min. Then, the suspension of eggs was sieved using a 20 μm sieve. Released eggs were collected in 5 mL of water, and the number of eggs was counted with the aid of an Olympus BX51 optical microscope .Four main treatment methods were included: (1) without any treatment (CK); (2) inoculation with strain PC-170 (PC); (3) inoculation with M. incognita and the right was not treated (M–O); (4) the left was pre-inoculated with PC-170 and challenged with M. incognita (PC+M–O); and (5) the left was pre-inoculated with PC-170 and the right was challenged with M. incognita (PC–M). The tomato variety used in the experiment was Lichun. All plants were placed in a completely random design, with 30 plants per treatment. Plant height and root and shoot weight were measured after 45 days. At the same time, the severity of nematode infection of plants was determined via the methods described above. Among them, treatments (3) and (5) were observed at 15 days, 25 days, and 35 days. At six points in time , root material was collected from tomato plants that underwent treatments (3) and (5), and then stored at −80 °C.The roots of tomato seedlings were evenly transplanted into two adjacent nutrient bowls, each of which was filled with soil mixed with 20 mL of PC-170 spore liquid. The split-root experiments consisted of five main treatments: (1) the left and right root systems were not treated (O–O); (2) the left was pre-inoculated with PC-170, but the right was not treated (PC–O); (3) the left was challenged with def1 mutant tomatoes with 20 mL of 0.1 mM methyl jasmonate (MeJA) solution. MeJA solution was prepared from a 100 mM MeJA (Sigma Odridge (Shanghai) Trading Co., Ltd., Shanghai, China) stock solution in 96% (v/v) ethanol. The experiment consisted of three main treatments: (1) half of the root system was challenged with M. incognita; (2) the left was pre-inoculated with PC-170 and the right was challenged with M. incognita; and (3) the left was pre-inoculated with PC-170 and treated with MeJA, while the right was challenged with M. incognita. All plants were placed in a completely random design, with 30 plants per treatment and three biological replicates for each treatment. At 1 h, 6 h, 12 h, 24 h, and 48 h after the first treatment with MeJA, and on the 10th day, the colonization of PC-170 in the roots was tested using the method described above. At 45 d, the severity of nematode infection of plants was determined via the methods described above.The split-root experiments described above were used for all mutant tomato lines. Then, we treated the 6 spores·mL−1 for use. Using the method of root irrigation, 15 mL of the spore solution prepared above was added to each tomato root. Tomato bacterial wilt was taken out of sterile distilled water, and then grown on TTC plates  for 36 h. Toxic colonies were transferred to the basic medium, and then sterile distilled water was added to prepare an inoculation suspension with a bacterial concentration of about 1 × 106 cfu mL−1 after expanding for 36–48 h. Cucumber mosaic virus (CMV) and tomato mosaic virus (ToMV) were inoculated by weighing 2 g of diseased leaves and then adding 10 mL of phosphate buffer to grind them thoroughly, and were then coated on the surface of tomato leaves that were lightly dusted with carborundum. The experiments were all carried out on Lichun tomatoes. Treatment was carried out on the 10th day after PC-170 inoculation. Each pathogen included two treatments: (1) Only pathogens were inoculated; (2) PC-170 was inoculated in advance before pathogens were inoculated. All plants were placed in a completely random design, with 30 plants per treatment. The test was repeated at least three times, and a consistent trend was obtained. The leaves of three plants were randomly collected and stored at −80 °C. Disease assessments of CMV and ToMV were conducted throughout the experiment. Specific disease severity ratings were carried out at 10 d and 20 d. Disease severity was measured according to previous research [Tomato fusarium wilt was grown on PDA plates with rifampicin. After a few days, it was inoculated in potato dextrose broth (PDB) medium and placed on a 28 °C shaker at 150 revolutions per minute (rpm) to collect spores. The spores were counted using a blood cell counter, and were then diluted to a final density of 1 × 10research . Diseaseresearch . The disresearch . Three rDisease incidence formula:PR-P6, PR1a, PAL and NPR1 was detected. The tomato pathogenesis-related protein (PR-P6) gene is a pathogenesis-related protein induced by SA, and a direct indicator of the SA response. The gene coding for pathogenesis-related protein (PR1a) is a common marker of SA-regulated responses [PAL) is the first enzyme in the phenylpropanoid metabolism pathway. Non-expressor of pathogen-related genes 1 (NPR1) was shown to be a key regulator of SA-mediated suppression of JA signaling [PI II, MC, LoxD, and OPR3 were detected. Genes encoding proteinase inhibitor II (PI II) and multicystatin (MC) are the typical JA marker genes [LoxD (lipoxygenase D) is involved in the early steps of the JA pathway, while OPR3 (12-oxophytodienoate 3 reductase) is a JA biosynthesis gene [To analyze the changes in the tomatoes’ SA and JA pathways as a result of the fungi–plant–nematode interaction, we examined the marker genes involved in the SA and JA pathways. In the SA pathway, the expression of the genes esponses . Phenylaignaling . Regardier genes ,50. LoxDsis gene ,48. PrimSlEF (X14449), and then the relative expression levels were calculated using the 2−ΔΔCT method.Total DNA was extracted using the Plant Genomic DNA Kit (TIANGEN BIOTECH (BEIJING) CO., Ltd.), while total RNA was extracted using the RNAprep Pure Plant Kit (TIANGEN BIOTECH (BEIJING) CO., Ltd.), according to the manufacturer’s instructions. First-strand cDNA was synthesized from 1 ug of total RNA using the FastKing gDNA Dispelling RT SuperMix (TIANGEN BIOTECH (BEIJING) CO., Ltd.). Real-time quantitative RT-PCR (qPCR) reactions and relative quantification of specific mRNA levels were performed using the Talent qPCR PreMix (SYBR Green) (TIANGEN BIOTECH (BEIJING) CO., Ltd.) according to the manufacturer’s instructions. Reactions were performed in triplicate in a Thermal Cycling StepOnePlus using the following profile: 95 °C for 15 min, 40 cycles at 95 °C for 10 s, 60 °C for 30 s and 72 °C for 60 s. The gene-specific primers are described in p < 0.05) between treatments. When the data met the normal distribution and the variance was homogeneous, a paired comparison using Student’s t-test was carried out, or means were compared using Tukey’s test (p < 0.05). When ANOVA assumptions were not met, multiple comparisons were conducted using the Kruskal–Wallis test, and groups separated by Dunnett’s test (p ≤ 0.05). All of the experiments were repeated at least three times, with similar results.All data were subjected to analysis of variance (ANOVA) using SPSS software  to determine significant differences  (spore concentration of 5 × 1010 spores·mL−1) with the highest concentration. The growth of tomato plants was observed and compared with the untreated natural growth control group; when the concentration of spore solution was 5 × 106 spores·mL−1, the growth of tomato plants was significantly promoted, while at high concentrations (spore concentration of 5 × 1010 spores·mL−1) tomato growth was inhibited (It has been reported that nhibited A.P. chlamydosporia at different timepoints during treatment (2). The concentration of PC-170 in the roots increased over time, but eventually reached a state of equilibrium  with PC-170.In order to determine the colonization process of PC-170 in tomato roots, we measured the total root colonization by ilibrium B. TherefMeloidogyne incognita-infected tomatoes exhibited visibly restricted growth; however, this phenomenon was alleviated in plants pre-inoculated with PC-170   B. Similaficantly C,D. Howe 10 daip E,F. Comp pathway G,H.Meloidogyne incognita, we observed the expression of each gene in the plants of the M–O treatment group, and found that the expression level of the PAL gene increased significantly at 1 days after inoculation with Meloidogyne incognita (daim). Compared with the expression level at 1 daim, the expression level decreased at 15 daim, then increased again at 30 daim, and decreased once more at 45 daim , dry root weight (DRW), fresh shoot weight (FSW), and dry shoot weight (DSW) were also significantly lower compared to those in the control group , the root galls of plants were reduced by approximately 50% in the PC+M−O treatment group, and by approximately 40.0% in the PC−M treatment group, compared with the control M−O treatment group. However, in SA-deficient 1 mutant A,B.PI II) gene expression was induced by exogenously applying methyl jasmonate (MeJA) to the roots. The induction of the PI II gene in plants pre-inoculated with PC-170 was stronger and more durable  def1 mutant tomatoes inoculated with PC-170 and treated with the exogenous hormone MeJA. We collected roots to detect the total root colonization after 10 days. Compared with the total colonization of PC-170 in Castlemart tomatoes, the total colonization of PC-170 in def1 mutant tomatoes increased significantly. However, the total colonization of PC-170 was reduced when def1 mutant tomatoes were treated with the exogenous hormone MeJA (We compared the colonization of PC-170 in the roots under the following different conditions: (1) Castlemart tomatoes inoculated with PC-170; (2) one MeJA .Plants, unlike other forms of life, cannot move to escape biotic or abiotic stresses. However, plants have evolved alongside microbes. The plant microbiome is a basic and important partner in protecting the plant from stresses, in that it not only synthesizes enzymes or metabolites and produces important phytohormones that can negatively affect plant pathogens and ensure the tolerance of the plant to environmental stress , but alsPochonia spp. can colonize roots; acting as a root endophyte, their capacity to colonize and proliferate in roots greatly varies between isolates [P. chlamydosporia has multiple functions in promoting plants’ growth and increasing their yield or promoting flowering when colonizing their roots [P. chlamydosporia isolate PC-170 colonizes and proliferates in tomato roots. Isolate PC-170 exerts a positive effect on tomato plant growth by promoting root development and increasing tomato growth under greenhouse conditions. Previous evidence also indicated that endophyte over-colonization could limit plant growth through an excess of nutrients transferred to fungal partners [PL-II was upregulated at 5 and 10 dai with PC-170. Interestingly, when using the PC-170 spore liquid inoculation method, low-concentration spore liquid grows slowly in the soil, but too high a concentration of spore liquid inhibits the growth of tomato plants. We speculate that PC-170 is a dose-dependent beneficial endophyte. In the JA-deficient def1 mutant (Castlemart) tomatoes, the effect of growth promotion by PC-170 was lost. Numerous reports showed that JA signaling was involved in carbohydrate partitioning and limiting colonization [def1 mutant (Castlemart) tomato plants, the colonization of PC-170 was not effectively controlled by the plant itself, leading to the inhibition of plant growth. Studies have shown that the colonization of P. chlamydosporia on barley roots can upregulate phytohormone-synthesis-related genes, such as auxin, ethylene, and jasmonic-acid-synthesis-related genes [It has been reported that isolates ,53,54. Sir roots  Our resupartners . An effepartners . Immune partners ,56. Likenization ,55,57. Tnization . In tomanization . Our resP. chlamydosporia, which is a nematode egg parasitoid fungus with worldwide distribution, has been applied to the biological control of nematodes in many economically important crops [P. chlamydosporia in lettuce, cucumbers, tomatoes, and bananas also revealed that it can reduce the infection rate of nematodes [nt crops ,13,14,15ematodes . In thisP. chlamydosporia plays an active role in suppressing plant pathogens. For example, colonization by P. chlamydosporia reduced the occurrence of other fungi in the roots [P. chlamydosporia was included. In Petri dish experiments, P. chlamydosporia reduced colonization of barley roots by Gaeumannomyces graminis var. tritici (Ggt), and also reduced necrotic symptoms [Many studies have proven that endophytic fungi are able to protect plants against a range of soil-borne pathogens. In studies of root endophytism, researchers found that a large proportion of fungal root endophytes are antagonistic to important root pathogens such as wilt and take-all fungi . P. chlahe roots . Some stsymptoms . In our Trichoderma atroviride (Ta) significantly reduces the number of galls and adult nematodes inside tomato roots. Ta induces systemic resistance against Meloidogyne javanica (Mj), as demonstrated by an in vivo split-root experiment, without the need for the organisms to be in direct contact [P. chlamydosporia in the fungus–plant interaction, study of split-root experiments demonstrates that the differential P. chlamydosporia isolates induce systemic resistance against M. incognita in tomatoes, but not in cucumber; thus, whether P. chlamydosporia can induce systemic resistance against root-knot nematodes depends on the plant species [P. chlamydosporia impacted the nematode parasitism in the split-root experiments, in a manner distinct from the possible direct nematicidal effects of the PC-170 fungus [P. chlamydosporia induced plant defense mechanisms in fungus–plant interaction studies. contact . In orde species . We also0 fungus . P. chlaTrichoderma species can protect plants from pathogens or insect attack by inducing defense responses [Trichoderma-induced resistance, and the response ability of plants inoculated with Trichoderma species against multiple pathogens was improved by the ability to alternately activate different resistance-regulatory mechanisms [Trichoderma priming of SA- and JA-dependent defenses in roots was plastic and adaptive to the parasitism stage. Trichoderma primed SA-regulated defenses to limit nematode root invasion, and enhanced JA-regulated defenses, which compromised galling and fecundity [P. Chlamydosporia colonization of roots [P. chlamydosporia-induced plant resistance by the split-root method revealed that the expression of the salicylic acid pathway (PR-1 gene) and the jasmonate signaling pathway (Lox D gene) in tomato roots was induced both after being inoculated with the fungal isolate and after nematode inoculation [P. chlamydosporia; after inoculation with nematodes, the relevant marker genes of the SA pathway were activated. We found that the SA and JA pathways exhibited a certain antagonism, but at a certain point in time showed a synergistic effect in the process of resistance to nematodes, by analyzing the expression of related genes of the two pathways at the stage of nematode growth and development, which is a plastic phenomenon similar to that revealed by research on the anti-nematode Trichoderma. During the period from the first infestation of nematodes to the occurrence of the second infection, both the SA pathway and the JA pathway are induced and activated. However, the colonization of PC-170 in the roots makes this induction more rapid and intense, so it can increase the resistance of tomatoes to nematodes. Kammerhofer [Heterodera schachtii, but during later syncytium and female development, salicylic acid seems to be a negative regulator. In the study of PC-170 inducing tomato resistance to pathogens, the activation of defense signaling pathways also occurred. When analyzing the expression of marker genes, it was found that different pathogens activate the SA and JA pathways in different orders, which is related to different pathogens’ different mechanisms of action on tomatoes. Among the pathogens to which PC-170 plays an active role in resistance, we found that the activation of the signaling pathway became more intense, and the fold and duration of gene upregulation were improved.esponses . There achanisms ,65. Tricecundity . In 2015of roots . Study oculation . In thismerhofer  found thT. harzianum was still able to protect the roots of the JA-deficient def1 mutant against nematode invasion, but was lost in SA-deficient NahG plants [T. harzianum was that we found that PC-170-mediated plant resistance against nematode invasion was restricted in NahG plants. Thus, we can conclude that the induction of SA-dependent defenses by PC-170 during M. incognita infection putatively contributed to plants’ resistance to nematodes. However, PC-170-mediated plant resistance to nematode invasion was completely blocked in def1 plants, while the JA-deficient def1 mutant could regain resistance to nematode infection in PC-170-pre-inoculated plants after spraying with the exogenous hormone MeJA; this is in contrast to the results of some other studies [We used mutant lines to verify the PC-170-induced resistance in tomatoes. In 2017, Martinez-Medina et al. showed that the systemic protection induced by G plants . We used studies ,37. The P. chlamydosporia colonizes and proliferates in tomato roots. Isolate PC-170 had a positive effect on tomato growth, promoted root development, and increased tomato growth under greenhouse conditions. PC-170 increased tomatoes’ resistance to root-knot nematodes and induced systemic resistance. The JA pathway of plants regulates the colonization of PC-170; then, the JA pathway cooperates with the SA pathway to induce resistance to M. incognita and other pathogens in tomatoes. One of the challenges for the future will be the establishment of assays to unravel the regulatory mechanism of endophytes, and to show how PC-170 orchestrates downstream responses during interactions with root cells.In conclusion, this study confirmed that


A high vaccination rate of older and particularly chronically ill people against coronavirus disease-2019 (COVID-19) is likely one of the most important factors in containing the pandemic. When Germany’s vaccination campaign started on December 2020, vaccination prioritization was initially carried out starting with older population groups. Side effect rates in 1065 individuals who had received the first dose of the messenger ribonucleic acid (mRNA) vaccine BNT162b2 Tozinameran from BioNTech/Pfizer three weeks earlier were examined retrospectively. An age- and gender-graded data analysis showed clear age and gender differences with regard to vaccine-related adverse effects. In 77% of all individuals over 80 years of age, no local or systemic side effects were reported after the first vaccination, whereas in the age group up to 80 years, only 37% showed no side effects. In the whole study population, 64% of females and 73% of males reported no adverse effects. The initial vaccination with mRNA vaccine BNT162b2 shows an overall low profile of side effects. Particularly in those over 80 years, an extraordinarily good tolerance with equally good effectiveness is evident. The sex comparison showed that women suffer more often from adverse vaccination reactions. In order to achieve sufficient herd immunity, both age- and gender-dependent vaccination reactions and any difference in the maintenance of immunity should be considered in future vaccination strategies. According to Johns Hopkins University, around 195 million people were infected with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus up to and including 28 July 2021 .Before a COVID-19 vaccine is administered, all candidates must be informed about possible vaccination reactions and given the opportunity to ask the provider of the vaccine questions. In addition, the candidate should be given an information sheet outlining the pertinent issues. The following non-age (or gender)-graded information on the BNT162b2 vaccine is documented in the information sheet of the Standing Vaccination Commission (STIKO) of the Robert Koch Institute: “The most frequently reported vaccination reactions in the approval studies were pain at the injection site (more than 80%), fatigue (more than 60%), headache (more than 50%), muscle pain and chills (more than 30%), joint pain (more than 20%), fever and swelling of the injection site (more than 10%).“ This may lead to fear and/or refusal to accept the vaccination, which is most pronounced in the age group of over 80 years . This vuThe goal of this work was to evaluate age- and gender-dependent frequencies of vaccination reactions after the first application of the mRNA COVID-19 vaccine BNT162b2 in order to make improved recommendations for future vaccination strategies.R) were evaluated. This group included 820 individuals over 80 years of age and 245 individuals younger than or equal to 80 years of age, who were entitled to be vaccinated in accordance with In a retrospective study of eight vaccination days in February 2021, the data from 1065 individuals at the vaccination center of Rhineland-Palatinate in Wissen  who had received an initial vaccination three weeks earlier with the mRNA vaccine BNT162b2 Tozinameran from BioNTech/Pfizer (trade name: ComirnatyThose that had received the first vaccination were given a standardized questionnaire about the side effects and complications that had occurred and the answers were ranked according to severity (1—mild to 5—severe) . These wThis retrospective study was carried out in accordance with the Helsinki Declaration and registered in the German Register for Clinical Studies . The prop values < 0.05 were considered to be statistically significant.The data were analyzed with IPM SPSS Statistics Version 26.0 . In the analysis, we assessed categorical differences (categories were gender or age groups) using the chi-square test and Pearson correlation, and additionally for individual parameters using the exact Fisher test. The Mann–Whitney U test was used for the non-normally distributed continuous variable age. In addition to the mean and standard deviation (SD), the median and the range were also included. In general, p < 0.001) from the number for the age group up to 80 years  (p < 0.001). With regard to the local vaccination reactions, 76% (105/139) in the age group up to 80 years of age and 86% (116/135) in the over-80-year-olds were only weakly pronounced with suggestive association (p = 0.077) (p = 0.717), an evaluation of the severity of the systemic side effects in the comparison of the ≤80-year-olds with the >80-year-olds was not meaningful -graded information sheet of the STIKO , as well 91/245) . In 43% = 0.077) . In the aningful .In the overall comparison of the age groups 20–39 years, 40–59 years, 60–80 years and >80 years, there was a tendency for the frequency of side effects  to increase with decreasing age , Table 3p < 0.001)  in the 20–39-year-olds, 42% (50/119) in the 40–59-year-olds, 57% (26/46) in the 60–80-year-olds and 84% (685/820) among the over-80-year-olds (< 0.001) , Table 3p < 0.001)  and 90% of the over-80-year-olds (737/820) showed no reactions to the vaccine, whereas the proportion of 40–59-year-olds  who showed no systemic vaccination reactions was comparable to the proportion of 60–80-year-olds without systemic side effects  (< 0.001) , Table 3p = 0.179) (The characteristics (mild/moderate/severe) of local and systemic side effects within the subgroups in relation to age differences showed a heterogeneous pattern without statistical significance (= 0.179) . Neitherp = 0.002)  (p < 0.001).With regard to a gender-graded data analysis, there was a significant difference. Women had significantly more side effects than men (= 0.002) . No reac= 0.012) . As in tEffective and well-tolerated vaccines to protect against COVID-19 are an important tool for containing the COVID-19 pandemic [Immunosenescence could provide an explanation for the phenomenon of the extremely high tolerability of the mRNA vaccine BNT162b2 in the elderly . The terThe safety profile was characterized by short-term, only mild to moderate local and systemic reactions in the pre-approval study for the BNT162b2 mRNA COVID-19 vaccine .It is intended that the side effect questionnaire provides accurate information on the pain perception of vaccinated individuals. With age, people seem to have a higher pain tolerance. A retrospective study reported that 22,963 patients who had undergone surgery in 105 German hospitals showed a linear decrease in postoperative pain intensity with increasing age for every type of surgical procedure . A studyR/VaxzevriaR), which has been associated with the very rare clinical picture of sinus thrombosis [In a current survey on COVID-19 vaccination readiness in Germany , 75%  in the United States, showing that 79.1% of the side effects after COVID-19 vaccination were reported by women, although they only represented 61.2% of the vaccinated population . The obsThe first vaccination with the mRNA vaccine BNT162b2 shows an overall moderate side effect profile. Age-graded data, especially in our focus group over 80 years of age, display very good tolerance. In a gender-specific analysis, our study also showed that females are more likely to experience side effects associated with the vaccine. In order to achieve sufficient herd immunity, age- and gender-dependent reactions to vaccination and different maintenance and duration of immunity should be considered in future vaccine development and administration strategies.


Rana chensinensis). Through the study of the structure and biological activity, it was found that Temporin-1CEh was a helical peptide from the Temporin family, and possessed good anti-Gram-positive bacteria activity through the mechanism of membrane destruction. Seven analogues were further designed to obtain broad-spectrum antimicrobial activity and higher stability in different physiological conditions. The results showed that T1CEh-KKPWW showed potent antibacterial activity with significantly increasing the activity against Gram-negative bacteria in vitro and in vivo with low haemolysis. In addition, T1CEh-KKPWW2 showed high sensitivity to the pH, serum or salts conditions, which applied a branched structure to allow the active units of the peptide to accumulate. Even though the haemolytic activity was increased, the stable antibacterial activity made this novel analogue meet the conditions to become a potential candidate in future antimicrobial and antibiofilm applications.Amphibian skin secretion is an ideal source of antimicrobial peptides that are difficult to induce drug resistance to due to their membrane-targeting mechanism as a new treatment scheme. In this study, a natural antimicrobial peptide Temporin-1CEh was identified by molecular cloning and mass spectrometry from the skin secretions of the Chinese forest frog ( Nowadays, the occurrence of multiple drug resistance of pathogens is increasing, and even some pathogens can evade the treatment of all clinically used antibiotics . The sevRana chensinensis, was used as the research object. Its dried skin, possessing anti-inflammation activity, as well as oviductus ranae are traditional Chinese medicines [Rana chensinensis have antibacterial, anticancer, antioxidant and anti-apoptotic activities [Even so, few AMPs have been approved for clinical use due to considerable drawbacks on specific pathogens, significant haemolysis, high production costs, susceptibility to protease degradation, and decreased activity in the presence of physiological concentrations ,9. A baledicines ,15. In ativities ,17,18,19Rana chensinensis frogs were obtained from commercial sources and the skin secretion obtaining process was described previously [Three adult eviously . The stuRana species  were used in a segment of the 5′-untranslated region. The degenerate primer was designed relating to the 5′-untranslated high conserved region of Rana-species.The shot-gun cloning was performed to isolate the prepropeptide encoding RNA, as described before . In the Temporin-1CEh and seven analogues were synthesised by using a Tribute automated solid-phase peptide synthesiser , as described previously . Fmoc-Ly2O. The resulting dispersions were extruded through a stack of two polycarbonate filters  using a Liposofast low pressure homogeniser  to obtain large unilamellar vesicles after five frozen-thaw cycles at 37 °C were performed. The concentration of total phosphorus was determined by the ascorbic acid/ammonium molybdate method, and 3 mM of liposomes was subjected to CD analysis [Appropriate amounts of dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidylglycerol (DOPG)  were dissolved in chloroform to obtain stock solutions. The desired compositions were mixed and then dried in a high vacuum evaporator until the lipid layer was formed at the bottom of the bottle. The lipid residues were subsequently hydrated at 50 °C with Hanalysis ,24.2O  (simulated comparably negatively charged environment of prokaryotic membrane), TFE/H2O   and H2O, respectively.The secondary structure of the peptides was examined by circular dichroism spectropolarimeter  and a quartz cuvette with a 1-mm path length following the previously described parameters setting . Each saS. aureus (ATCC CRM 6538), MRSA (NCTC 12493) and E. faecalis (NCTC 12697), Gram-negative bacteria, E. coli (NCTC 10418), P. aeruginosa (ATCC 27853) and K. pneumoniae (ATCC 43816). A measure of 5 × 105 CFU/mL of bacterial suspension including 2-fold dilution peptides (ranging from 1 µM to 512 µM) was incubated in 96-well plates for 16–20 h at 37 °C. For the positive and negative control, 200 mg/L of final concentration of norfloxacin and 1% DMSO were applied, respectively. The optical density of each well was determined at 550 nm using a Synergy HT plate reader  and the lowest concentration of peptide that resulted in no growth of the bacteria was defined as the MICs. Subsequently, 10 µL of the solution in each clear well was dropped on a Mueller–Hinton agar (MHA) plate and incubated at 37 °C for 16–20 h. The lowest concentration without colony growth was considered as the MBCs.ESKAPE pathogens, encompassing six pathogens which exhibit multidrug resistance and lead nosocomial infections, were used to determine the antibacterial activity of Temporin-1CEh and its analogues by using broth dilution method , includiS. aureus and E. coli, as indicated previously [The anti-biofilm activities were tested on eviously . For theS. aureus and E. coli, the SYTOX-GREEN nucleic acid stain was used in the assay indicated before [S. aureus and E. coli, respectively.In order to test the permeability percentage of Temporin-1CEh and its analogues against d before . Tryptic2 and 4 µM of FeCl3, respectively, were prepared. In particular, the pathogens were diluted by the preprepared mediums to a final concentration of 5 × 105 CFU/mL after growing to the logarithmic phase, and other operations followed the MIC assay.Mueller–Hinton broth (MHB) at pH 6.0 and 8.0 as well as MHB containing 10% of Fetal Bovine Serum (FBS), 150 mM of NaCl, 1 mM of MgClv/v) of the non-ionic detergent, Triton X-100  in PBS solution, were used as negative and positive controls, respectively. HC50 was defined as the mean peptide concentration producing 50% haemolysis.The erythrocytes from defibrinated horse blood  were used to perform the haemolysis assay, as described previously . Equal vGalleria mellonella) were used as the infection model. The larvae  were selected between 225 and 275 mg (9 larvae per group in a Petri dish). For the positive and negative controls, 50 mg/kg of vancomycin (25 mg/kg of ampicillin for E. coli) and PBS were employed. Final concentration of S. aureus and E. coli cells injected was 5 × 105 and 5 × 106 CFU/larva, respectively. The assay was performed as described before [In this assay, the larvae of the wax moth (d before  and the p < 0.05), ** (p < 0.01) and *** (p < 0.001).Data were subjected to statistical analysis using Prism . Error bars in the graphs represent the standard error of the mean (SEM) with experiments performed on more than three sets of replicates. The significance was determined using Mantel–Cox test for survival curve of wax moth larva, indicated by ns (none significant difference), * (Rana chensinensis is shown in The sequences of nucleotide and translated open-reading frame (ORF) amino acids of the precursor cDNA encoding the peptide Temporin-1CEh from the skin secretion library of Rana chensinensis. The RP-HPLC chromatography of the skin secretions of Rana chensinensis, annotated MS/MS spectrum of Temporin-1CEh and MS/MS fragmentation sequencing analysis, are shown in With the use of RP-HPLC and mass spectrometry followed later by analysis using MS/MS fragmentation sequencing, Temporin-1CEh was identified in the skin secretion of 50 of Temporin-1CEh was 152.6 μM compared to that of Temporin-1CEa which was 160 μM [All sequences of peptides are shown in s 160 μM . SeveralFirstly, decreasing the haemolytic activity of Temporin-1CEh was the first goal. Four amino acids including isoleucine and phenylalanine at the C-terminus of Temporin-1CEh and Temporin-1CEa were removed in order to decrease the hydrophobicity obtaining T1CEh-t and T1CEa-t. This similar operation in a previous study had ensured the helical structure integrity of the central area . IsoleucE. coli, as well as maintained the low level of haemolysis.Secondly, two lysines were added onto the N-terminus to increase the antimicrobial activity against Gram-negative bacteria (T1CEh-KK). This strategy has been used in the modification of Temporin B in a previous study . With thThirdly, considering the proline at position 3 is a conserved motif in the Temporin peptide family , the aspE. faecalis, the minimum inhibitory concentration (MIC) of these two peptides ranged from 4 to 32 μM. The haemolysis rates of these two peptides were lower than 6%.What is more, according to a previous study, the indole side chain of tryptophan has a unique interaction with the polar-nonpolar interface, making the peptide anchored in the membrane interface region . PhenylaE. faecalis, as well as a twofold decrease against P. aeruginosa and E. coli, comparing with that of T1CEh-KKPWW. Even though the haemolytic activity of T1CEh-KKPWW2 experienced an increase comparing with T1CEh-KKPWW , the HC50 of T1CEh-KKPWW2 was 569.8 μM which was higher than that of Temporin-1CEh.Finally, the lysine which the side chain protected by the Fmoc group was applied in peptide synthesis to build a peptide with a branched structure. Compared with monomer peptides, branched peptides showed higher bioactivity due to the local concentration of bioactive units being increased ,32. In aS. aureus (DOPC:DOPG = 1:1) and E. coli (DOPE:DOPG = 3:1) liposome constitutions  spectra showed, all peptides formed random coil structures in aqueous environments . Howeveritutions . TemporiS. aureus and E. coli  against  E. coli . T1CEh-KS. aureus (ATCC CRM 6538) and MRSA (NCTC 12493) were employed to study the ability of Temporin-1CEh, T1CEh-KKPW, T1CEh-KKPWW and T1CEh-KKPWW2 to inhibit biofilm formation and eradicate mature biofilm. Four peptides inhibited 90% of biofilm production of S. aureus and E. coli from 8 to 64 μM  might cause the competitive inhibition to the binding between cationic peptides and bacterial membranes [In a previous report, high concentrations of salts, acidic pH and serum could cause a decrease or loss of antimicrobial activity for AMPs . At the embranes .In order to improve the antibacterial ability of T1CEh-KKPWW in different physiological conditions, a branched structure was used as a modification strategy obtaining peptide T1CEh-KKPWW2. T1CEh-KKPWW2 showed a slight decrease in the antimicrobial activity against ESKAPE pathogens, showing only two- to four-fold weakening of the MIC values. This improvement allowed T1CEh-KKPWW2 to prevent the cation competitive inhibition comparing with T1CEh-KKPWW. It was noticed that the permeability of T1CEh-KKPWW2 at high concentrations showed decreasing trends, which might be due to the binding between peptides and nucleic acids. Previous studies have shown that the peptide LL-37 prevented the degradation of DNA from NETs, purified neutrophil and calf thymus cells derived from bacterial pathogens . In partIn addition, the phenomenon of aggregation can be found in T1CEh-KKPWW2 at a high concentration, achieving hydrogel. The aromatic stacking which may cause complicated π−π interactions played a role in the self-assembly in many amyloid-related proteins ,53. At tThe application of branched structure most directly leads to the accumulation of the functional unit to the peptide. Net charges and the hydrophobicity increase of T1CEh-KKPWW2 not only promoted its antimicrobial and antibiofilm activity, but also increased its haemolysis. The same situation could be found in the bioactivity evaluation between branched peptide SB056 and its linear peptide . HoweverGalleria mellonella larvae model demonstrated the potential antimicrobial efficacy of selected peptides in vivo [Staphylococcus activity in vivo, and all treatment groups exhibited statistical differences compared with the negative control group. However, the reason for no improvement of mortality with increasing doses is not clear. It might be related to the rapid degradation of peptides in vivo. In addition, T1CEh-KKPWW2 showed better anti-Escherichia activity in vivo than anti-Staphylococcus activity in vivo.Additionally, the  in vivo . TemporiIn summary, we reported the study on the characterisation of a novel temporin peptide, the design of the N- and C-terminal domains modified analogues and assessed the influence on their bio-efficacy with in vitro and in vivo models. It revealed that increasing the net charge at the N-terminal domain could improve the selectivity, whilst the hydrophobic interaction with amino acid residues ensures the membrane permeabilisation. The balance of both features is recognised as a key that could be favourable for developing the new therapeutic approaches in the treatment of infectious diseases.


Carbon black (CB), carbon nanotubes (CNTs), and graphene nanoplatelets (GnPs) individually or doubly served as reinforcing fillers in polycarbonate (PC)/poly(vinylidene fluoride) (PVDF)-blend (designated CF)-based nanocomposites. Additionally, organo-montmorillonite (15A) was incorporated simultaneously with the individual carbon fillers to form hybrid filler nanocomposites. Microscopic images confirmed the selective localization of carbon fillers, mainly in the continuous PC phase, while 15A located in the PVDF domains. Differential scanning calorimetry results showed that blending PVDF with PC or forming single/double carbon filler composites resulted in lower PVDF crystallization temperature during cooling. However, PVDF crystallization was promoted by the inclusion of 15A, and the growth of β-form crystals was induced. The rigidity of the CF blend increased after the formation of nanocomposites. Among the three individually added carbon fillers, GnPs improved the CF moduli the most; the simultaneous loading of CNT/GnP resulted in the highest moduli by up to 33%/46% increases in tensile/flexural moduli, respectively, compared with those of the CF blend. Rheological viscosity results showed that adding CNTs increased the complex viscosity of the blend to a greater extent than did adding CB or GnPs, and the viscosity further increased after adding 15A. The electrical resistivity of the blend decreased with the inclusion of carbon fillers, particularly with CNT loading. Polymer blends and composites are fabricated to improve the specific properties of parent polymer components for broadening their applications. Blend/composite products have continuously attracted great attention from academia and industry. The miscibility, phase morphology, and type/dispersibility of added fillers are the key factors in controlling the final properties of polymer blends and composites. Polymer-based nanocomposites have received more interest than conventional polymer blends/composites in the last two decades ,2. The pBisphenol A-type polycarbonate (PC) is a well-known engineering plastic. It has diverse applications in the car, medicine, and electronics industries due to its high glass transition temperature, superior mechanical properties, transparency, and flame retardancy. PC-based nanocomposites have been prepared and studied. Hsieh et al.  successfPoly(vinylidene fluoride) (PVDF) is an important engineering plastic possessing unique dielectric properties, excellent thermal/chemical stability, and other advantageous properties . Among iHitherto, various combinations of polymer/nanofiller binary nanocomposite systems have been successfully fabricated and characterized. Recently, polymer-blend-based multicomponent nanocomposites have attracted great attention due to their potential in advanced applications ,29,30,31According to the literature, nanocomposites fabricated with a combination of polymer blends and hybrid nanofillers have seldom been investigated. To broaden the applications of polymer blends, the individual and simultaneous loading of diverse nanofillers on the resultant properties is worthy of systematic investigation. In a previous study, PVDF/PC-70/30-blend-based nanocomposites were prepared and characterized . In the Bisphenol A-type PC  with a melt flow rate of 30 g/10 min was used as the main component for preparing samples. PVDF  was used to blend with PC to form a PC-based blend. Commercially available carbon nanofillers, multiwalled CNTs, GnPs, and carbon black (CB), were used for the preparation of composites. The CNTs  had a carbon purity of >99% and an average outer diameter of 60 nm. The GnPs  possessed high purity (>99.5%) and a thickness of about 6–8 nm. The CB  had an average diameter of 30 nm. In addition to the three different carbon nanofillers, commercial O-MMT  was used as the extra nanofiller for the preparation of hybrid filler composites. Cloisite 15A is reported to have been modified with ca. 40% of dimethyl dihydrogenated tallow quaternary ammonium ion, and the tallow is composed of ca. 65% C18, 30% C16, and 5% C14. The cation exchange capacity of 15A is 125 mequiv/100 g.PC/PVDF-blend and blend-based composites were melt-mixed using a Haake PolyDrive internal mixer (R600) with a Banbury-type rotor running at 60 rpm. Prior to mixing, the components were dried in an oven  at 70 °C for 24 h to remove absorbed moisture. The blend and composites were designed to have a PC-PVDF weight ratio of 7 to 3. The components  were mixed at 240 °C for 13 min. PC and PVDF components were first mixed for 5 min, and then the nanofiller(s) was incorporated with mixing continued for 8 min. The sample code of CF denotes the PC/PVDF blend, and CF-B, CF-T, CF-P, CF-BA, CF-TA, CF-PA, CF-TP, CF-TB, and CF-BP represent 1 wt.% of the different nanofillers loaded into the blend. B, T, P, and A denote CB, CNTs, GnPs, and 15A, respectively. For example, CF-T is the composite with 1 wt.% CNT, and CF-PA is the composite with 1 wt.% each of GnPs and 15A. For comparison, neat PC and PVDF were melt-treated under the same mixing conditions.2 using a TA DMA Q800  system with measurements conducted in single cantilever mode over the temperature range of 25–150 °C at a 2 °C/min heating rate and a frequency of 1 Hz. An Anton Paar Physica rheometer  in oscillating mode at 1% strain amplitude was utilized to assess the complex viscosity of the samples at 240 °C. The experiments were conducted with a parallel plate geometry (25 mm diameter and 1 mm gap), and the sweep frequency was in the range of 0.1–100 rad/s. The surface electrical resistivity of compression-molded specimens was measured using MCP-HT450 resistivity meters . The reported values are the average of five repeated measurements obtained at various locations of each prepared strip.Phase morphology and nanofiller(s) dispersion of the samples were assessed by a scanning electron microscope (SEM) combined with an energy dispersive spectroscope . The compression-molded samples were fractured after immersion in liquid nitrogen and coated with gold prior to observation. A Hitachi S-3000N SEM  was employed to conduct the experiments, and an “ImageJ” software was used to determine the domain size of the dispersed phase in the fractured blend/composites. An X-ray unit  with CuKα radiation (λ = 1.54 Å) was used to examine the PVDF crystal structure of the samples. The XRD was operated at 30 kV and 20 mA, and the compression-molded film samples were tested with a 2θ scan of 1.2°/min and a step size of 0.02°. A TA DSC Q10  analyzer was employed to assess the crystallization and melting behavior of PVDF in different samples. For the crystallization study, samples of ca. 5 mg were first melted at 240 °C and then cooled to 20 °C at different rates. The precooled samples were afterwards scanned at 20 °C/min to 240 °C to evaluate the melting of PVDF. A thermogravimetric analyzer  was employed to study the thermal degradation behavior of the samples (ca. 10 mg). The samples were heated from room temperature to 680 °C at 20 °C/min in a nitrogen environment. The tensile modulus (TM) and flexural modulus (FM) of specimens prepared by compression molding (according to ASTMD638/ASTMD790) were determined at a strain rate of 2 mm/min and 1 mm/min, respectively, with a Gotech Al-3000 system . The reported TM/FM values of each formulation are averages of five specimens. The storage modulus (SM) of compression-molded specimens (30 mm × 6 mm × 0.4 mm) was measured in NTo clearly reveal the localization of added nanofiller(s) in the blend matrix, XRD patterns of neat components and the CF blend cooled at 5 °C/min from the melt state are illustrated in p, at the exotherm minimum) of neat PVDF was around 139 °C, and it shifted noticeably to a lower temperature (main peak) of ca. 107 °C after CF blend formation. The evident shift should be attributed to the homogeneous nucleation of PVDF in the various small PVDF domains in the blend [p value (ca. 129 °C) evidently higher than that of the CF blend. This observation might be ascribed to the large dimensions of GnPs inducing less homogeneous nucleation in PVDF domains. That is, GnPs had somehow contacted the PVDF domains, and then led to a minor nucleation effect for PVDF. For the 15A-included (hybrid filler) composites, the crystallization of PVDF appeared at higher temperatures compared with that of neat PVDF. The higher-temperature PVDF crystallization could have stemmed from the 15A-nucleated/induced growth of α- and β-form crystals, as 15A was distributed in the PVDF domains. For the hybrid two carbon nanofiller composites, the GnP-added ones showed a main peak close to that of CF-P. The GnPs-accelerated PVDF crystallization in the composites was again demonstrated. The main Tp values of the samples are compared in The effects of blending with PC and adding different nanofillers on the crystallization of PVDF were investigated through DSC. he blend . The comm) at 167–169 °C. The transition around 147–152 °C of each PC-included curve was the glass transition of PC. An evidently lower-temperature endotherm (arrowed) partially overlapping the main melting peak was additionally seen for CF-P and CF-TP samples. The two meltings of PVDF were associated with the individual melting of originally grown and heating-process-recrystallized α-form crystals. The evident lower temperature melting observed in CF-P/CF-TP suggests that GnPs must have caused the growth of more stable original crystals compared with CB and CNTs. As a consequence, the melting of original crystals could be observed along with the melting of heating-recrystallized, more stable crystals. That is, a less amount of crystals grew during the heating scan of CF-P/CF-TP. The relatively higher crystallization temperatures  of CF-P/CF-TP  than that of neat PVDF was observed. Per XRD data, a higher Tm could be associated with the melting overlap of 15A-induced β-form crystals and some α-form crystals [ms of the samples are summarized in -P/CF-TP  are respcrystals . The maid5, mainly PVDF degradation) and 50%  weight loss for the individual samples are compared in 13 Ω-cm, indicating their electrically insulating nature. The individual addition of 1 wt.% CB (CF-B) and 1 wt.% CNTs (CF-T) reduced the resistivity by 3–4 orders of magnitude to about 1010 and 109 Ω-cm, respectively. For the 1 wt.% GnP composite (CF-P), the ER dropped only about 2 orders, from 1013 to 1011 Ω-cm. The different degrees of drop in ER after adding various carbon nanofillers are mainly attributed to the combined effects of aspect ratio and dispersion of the individual nanofillers. The size- and dispersion-related surface area of individual carbon nanofillers also played a role in influencing the resultant property [8 Ω-cm, only 1 order lower compared with CF-T. Based on the result, it is expected that the CF blend with 2 wt.% CNT inclusion will exhibit lower ER than CF-TB . The percolation behavior of the single and double carbon filler composites will be investigated in a future study.The addition of conductive nanofiller(s) to the polymer matrix may lead to (semi)conductive products. The surface electrical resistivity (ER) of the samples was measured, and values are listed in property . CNTs shThe dispersion status of individually and simultaneously added nanofillers  in a PC/PVDF-70/30 blend was evaluated. The achievement of single and hybrid filler nanocomposites was confirmed. The carbon fillers were mainly localized in the major PC phase, while 15A located in the dispersed PVDF domains. Blending with PC caused an evidently lower crystallization temperature of PVDF (due to the homogeneous nucleation), whereas adding 15A promoted PVDF crystallization and induced the formation of higher melting temperature β-form PVDF crystals (XRD result) in the hybrid filler nanocomposites. The addition of CB and/or CNTs barely changed the crystallization and melting behavior of PVDF in the blend, while adding GnPs modified those properties. The positive effect of GnPs on the thermal stability of the PC/PVDF blend was demonstrated, whereas CB and CNTs hardly had any influence. Adding a layered 15A increased the degradation temperature of PC in the composites. The rigidity  of the blend increased after adding carbon nanofillers, and GnPs exhibited higher efficiency than CB and CNTs. The effectiveness of carbon nanofillers in increasing the storage modulus also followed the sequence GnP > CNT > CB. A slight decrease in rigidity was noticed when 15A was added to the single carbon filler composites. The double carbon filler composites exhibited higher rigidity compared with the single carbon filler systems; the coexistence of CNTs and GnPs exhibited the highest rigidity among the samples tested. An increase in complex viscosity was observed for the nanocomposites, suggesting that a pseudo-network structure was developed. CNTs led to a more evident increase compared with CB and GnPs; further addition of 15A increased the viscosity. The reduction in the electrical resistivity of the PC/PVDF blend followed the sequence of adding CNT > CB > GnP. The addition of 1 wt.% CNTs reduced the electrical resistivity by 4 orders of magnitude; the coexistence of 1 wt.% individual CNTs and CB dropped the resistivity the most, up to 5 orders. The presence of 15A increased the electrical resistivity of the single carbon filler composites.


Stroke is among the leading causes of death worldwide, and stroke patients are more likely to live with permanent disabilities even after treatment. Several treatments are being developed to improve the quality of life of patients; however, these treatments still have important limitations. Our study thus sought to evaluate the neural differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) at various pulsed electromagnetic field (PEMF) frequencies. Furthermore, the effects of selected frequencies in vivo were also evaluated using a mouse ischemia stroke model. Cell proliferation decreased by 20% in the PEMF group, as demonstrated by the 3--2,5-diphenyl tetrazolium bromide (MTT) assay, and lactate dehydrogenase (LDH) secretion increased by approximately 10% in an LDH release assay. Fluorescence-activated cell sorting (FACS) analysis demonstrated that CD73 and CD105 were downregulated in the PEMF group at 60 Hz. Moreover, microtubule-associated protein 2 (MAP-2) and neurofilament light chain (NF-L) were upregulated in cell cultures at 60 and 75 Hz. To assess the effects of PEMF in vivo, cerebral ischemia mice were exposed to a PEMF at 60 Hz. Neural-related proteins were significantly upregulated in the PEMF groups compared with the control and cell group. Upon conducting rotarod tests, the cell/PEMF group exhibited significant differences in motor coordination at 13 days post-treatment when compared with the control and stem-cell-treated group. Furthermore, the cell and cell/PEMF group exhibited a significant reduction in the expression of matrix metalloproteinase-9 (MMP-9), tumor necrosis factor-alpha (TNF- Despite current declines in the mortality rates associated with neurological diseases, stroke continues to be the second-leading cause of death and long-term disability worldwide ,2. StrokSeveral studies have successfully employed pulsed electromagnetic field (PEMF) technology as an alternative non-invasive treatment method in a variety of fields. Particularly, electromagnetic fields (EMFs) have been found to enhance peripheral nerve regeneration  in rat models; increase myofibroblast populations; contribute to wound closure during diabetic wound healing ; and promote chondrocyte proliferation, matrix synthesis, and chondrogenic differentiation in rat models ,15. AnotThe purpose of this study was to identify the optimal PEMF frequency for neurodifferentiation at various intensities in vitro, and to evaluate the wound-healing efficacy in a mouse stroke model. Additionally, to gain insights into the poststroke wound healing process, our study assessed whether stem cells and electromagnetic fields could synergistically aid in the treatment of ischemic stroke.The hBM-MSCs were cultured in a neural-induction medium and exposed to PEMF . After tThe expression levels of CD73 and CD105 were evaluated three days after culture using FACS analysis. In the control group, the expression levels of CD73 and CD105 were 92% and 83%, respectively. As shown in As shown in After three days of culture, the expression of neural-related markers at the mRNA and protein levels were quantified via RT-PCR and Western blotting analyses. The mRNA expression levels of the neural development genes Neuro D1, MAP-2, Tau, MBP, DCX, and NF-L were determined using total mRNA obtained after three days. As shown in To assess whether PEMF treatment affected the behavior and motor coordination of mice with cerebral ischemia, a rotarod test was conducted daily after hBM-MSC transplantation. All data are presented as a percentage relative to the rotarod results of normal animals before treatment. The graph in All mice were sacrificed after 15 days of treatment and endured the treatments without significant weight loss. The expression levels of the neural development proteins Tau, Neuro D1, and Nestin in brain tissue were evaluated by Western blotting and the ImageJ software. There was a significant increase in the expression level of neural proteins in the cell group and the cell/PEMF group compared to the control group . In the Cerebral infarction lesions in brain tissues were confirmed via H&E staining. Prussian Blue staining was conducted to confirm that the injected cells had successfully migrated to the damaged brain area. As illustrated in In this study, neural differentiation of hBM-MSCs was induced via PEMF treatment to assess whether this could serve as a therapy to aid in poststroke recovery, as well as to demonstrate the effect of electromagnetic fields on the post-transplantation healing effects of hBM-MSCs in mouse ischemic models. First, we examined the cell morphological changes and toxicity of neural-induced hBM-MSCs after exposure to various PEMF intensities. Previous studies have comprehensively characterized the effects of neural differentiation media on hBM-MSCs ,20,21,22FACS was then performed to assess the differentiation of mesenchymal stem cells (MSCs) . Low CD7Our results also confirmed the neural differentiation of hBM-MSCs by characterizing the expression of mRNA and proteins as shown in This study evaluated the effect of PEMF on the phosphorylation of CREB and ERK through Western blotting B. Both tAs shown in As shown in Cytokines play an important role in the communication between cells, and they regulate survival, growth, differentiation, and effector functions . MMPs ar2 humidified atmosphere. The culture medium was changed twice a week. The cells were passaged before reaching confluence and were used after five passages. To induce neural differentiation, the cells were cultured in neural-induction media with a formulation based on previous publications with some modifications [The hBM-MSCs were purchased from ATCC (ATCC PCS-500-012). The cells were cultured in a nonhematopoietic (NH) stem cell medium  containing 1% penicillin/streptomycin  at 37 °C in a 5% COications . The neu2 humidified air.The PEMF was generated using a pair of Helmholtz coils. This device produced a region of nearly uniform magnetic fields. The stimulation unit was designed to handle a pair of identical coils 30 cm in diameter assembled in a Helmholtz configuration. A continuous PEMF  was used in this study. Alternating current was used to generate the electromagnetic fields with the coils. The current in the coil was generated using the COMSOL 3.4 simulation model , and the intensity was measured using a TM-701 Gaussmeter . The PEMF system was placed in an incubator  at 37 °C in 5% CO4 cells/mL) were then placed in the base of the PEMF system. The control cells were incubated under the same experimental conditions as the group without the PEMF. Changes in the cell morphology under different culture conditions were observed using a microscope . Cell morphology photographs were taken three days after PEMF exposure.The Helmholtz coil was placed in the cell incubator, and the field was set to various continuous PEMF frequencies, including 30 Hz, 45 Hz, 60 Hz, and 75 Hz, each with a field intensity of 10 mT, for 30 min per day for three days. The hBM-MSCs (1.0 × 104 cells/mL) were incubated in 0.5 mg/mL MTT-supplemented cell culture medium at 37 °C and 5% CO2 for 2 h. All experiments were performed in triplicate (n = 3). The purple-colored formazan derivative formed during the active cell metabolism was eluted and dissolved in 1 mL dimethyl sulfoxide . The absorbance was measured at a 570 nm wavelength. LDH activity was measured using an LDH-LQ kit . After three days of culture, 20 μL medium aliquots and 50 μL of working solution were thoroughly mixed and incubated in the dark at room temperature for 30 min. The reaction was terminated through the addition of a stop solution (1N HCl), and the absorbance was measured at a 570 nm wavelength.Cell growth was determined via the MTT assay after three days of cell culture coupled with PEMF exposure. The cells (1.0 × 10Cells were characterized by using a fluorescence-activated cell sorting (FACS) assay after three days of cell culture with PEMF exposure. CD 73 monoclonal antibodies  and CD 105 (Endoglin) monoclonal antibodies  were used to detect human antigens. Cells were incubated with fluorescein isothiocyanate (FITC) or with 200 μL of PBS and phycoerythrin (PE)-conjugated antibodies for 20 min at room temperature. The fluorescence intensity was examined using a flow cytometer , and the data were analyzed using the CellQuest software (BD Biosciences).Total cellular RNA was extracted using the TRIzol reagent  according to the manufacturer’s instructions. Total RNA concentrations and purity were measured using a NanoDrop spectrophotometer . A total of 2 μg of RNA was used for cDNA synthesis. Reverse transcription (RT) was conducted using the Advantage RT-PCR kit . Primers for RT-PCR were purchased from Bioneer  for the following genes: GAPDH, Neuro D1, MAP2, Tau, MBP, DCX, NF-L, Wnt, and β-catenin. All primer sequences are summarized in n = 30). The experimental protocol was approved by the Institutional Animal Care and Use Committee of Dongguk University (IACUC-2019-023-1). To create the stroke model, ischemic surgery was conducted as described by Choi et al. [−1 day−1). The lesioned animals were then randomly assigned to different experimental groups: control group , cell group , and cell/PEMF group . Fifth passage hBM-MSCs were used for transplantation, and these cells were implanted into the mice without inducing neuronal differentiation. Neural-induced hBM-MSCs were also identified in vitro . Animal testing was conducted under low-frequency conditions to ensure animal safety. The mice were exposed to PEMF for 30 min at a 10 mT intensity and a 60 Hz frequency. The hBM-MSCs were administered by inserting magnetic nanoparticles into the cell cultures to evaluate their movement and location in the animal prior to injection. Water-dispersible and biocompatible magnetic iron oxide (Fe3O4) nanoparticles (MNPs) were prepared as described in a previous study [5 cells) including MNPs were injected in saline via the penis, and the cell/PEMF group was exposed to PEMF  for 30 min per day for 15 days.Experiments were performed using C57BL/6 male mice  , 2% SDS, and 10% glycerol in Tris-HCl, pH 6.8 , and boiled at 100 °C for 5 min. The protein concentrations of total lysates were determined via the BCA Protein Assay . 2-Mercaptoethanol  was added to the sample buffer to a 10% concentration. The same amount of denatured protein (20–40 μg) was resuspended in a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane. Membranes were blocked with 5% skim milk in TBS-T (pH 7.4) for 30 min at room temperature, followed by overnight incubation with antibodies at 4 °C. The following primary antibodies were used: β-actin, p-ERK, β-catenin , and p-CREB . Antibodies were appropriately diluted in TBS-T containing 0.05% BSA. After washing, membranes were incubated with antimouse or antirabbit secondary antibodies  diluted in 5% skim milk in TBS-T for 2 h at room temperature. Band images were obtained using a ChemiDoc XRS+ gel imaging system  using ECL solution . Quantitative analysis of Western blot band images was conducted using the ImageJ software .Brain tissues were obtained using a biopsy punch 15 days after the animal experiments. A 3 mm diameter and 3 mm depth orifice were punched into the right front core, which is where the cerebral infarction region was made . β-actin, Tau , Neuro D1, and Nestin  were used as primary antibodies, and Western blotting was performed as described above.Cells on cover slides were fixed with 10% formalin for 30 min and washed with 10 mM Tris-HCl, pH 7.2. These cover slides were incubated with the respective monoclonal antibodies against anti-MAP-2  for 24 h, followed by development using the EnVision Plus reagent and Mayer’s hematoxylin as a counterstain. The hBM-MSCs were then fixed with 4% paraformaldehyde in PBS for 15 min. PBS solution containing 1.5% bovine serum albumin (BSA) was used for cell blocking. Cells were permeabilized using 0.5% Triton X-100 for 16 h at 4 °C mixed with the primary antibodies, which included anti-Neurofilament . The cells were washed in PBS for 10 min and incubated with antirabbit IgG-Alexa Fluor 488 conjugate or antimouse IgG-Alexa Fluor 555 conjugate  for 1 h at room temperature. The coverslips were counterstained with Vectashield mounting media containing 4‘,6-diamidino-2-phenylindole  for fluorescence and mounted on microscope slides. The cells were examined using a model LSM510-meta confocal laser scanning microscope .For hematoxylin and eosin (H&E) staining, the brain tissue was fixed in 4% paraformaldehyde for 72 h, dehydrated in ethanol, embedded in paraffin, and cut into 5 μm thick sections. For Prussian blue staining, brain tissues were washed with PBS and incubated in 5% potassium ferrocyanide in 5% hydrochloride acid. After 30 min, the slides were washed with PBS, counterstained, and examined under a light microscope. For immunohistochemical (IHC) staining, brain sections were incubated with anti-Neuro D1, anti-NF, anti-BDNF, anti-MMP-9, anti-IFN-γ, and anti-TNF-α antibodies . The sections were quenched with hydrogen peroxide, unmasked with hyaluronidase, and blocked in 10% normal donkey serum, then incubated with primary antibody for 1 h, washed, incubated with biotinylated secondary antibody, visualized with the Vectastain ABC staining kit , and developed in 3,3-diaminobenzidine.The rotarod test was used to measure motor coordination and fatigue prevention in mice after brain ischemia treatment. Only animals that were able to stay in the rotating rod for more than 180 s were selected for this study. The experimental mice were trained daily for three days before the surgery for the rotarod  test. To evaluate animal behavior, the rotarod test was performed every day for 15 days after transplantation. The mice were placed on the rotating rod at 15 rpm. The test consisted of three blind trials separated by 10 min intervals. The time spent on the rod was calculated after the mice fell from the test apparatus. t-test. The results of the animal tests were analyzed using a one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. A p-value < 0.05 was considered statistically significant.All data were analyzed using the SPSS 10.0 software  and are reported as mean ± SD. The differences between the groups were assessed using Student’s Stroke is among the leading causes of serious disability and death worldwide, and its occurrence has been rising for decades due to recent demographic shifts and aging populations. MSCs have been proven to be safe alternatives for stroke treatment. However, their efficacy as a poststroke therapy continues to be limited. Recently, PEMF (a non-invasive treatment that can be safely used in the long term) has been successfully used as a regulation therapy for a wide range of clinical conditions. In this study, physical PEMF treatment was used to minimize stroke side effects to improve the limitations of hBM-MSC therapy. Our findings confirmed that PEMF induced phosphorylation of ERK and CREB in vitro to promote hBM-MSC neurodifferentiation. Additionally, it was confirmed that the injected hBM-MSCs moved to the wound of the mouse stroke model. Using immunochemical staining, we confirmed that the injected cells could relieve inflammation of the ischemic area. In conclusion, we identified positive effects such as neurodifferentiation, motor function recovery, and nerve regeneration induced by inflammatory cytokine reduction in the PEMF-treated experimental groups with both in vitro and in vivo hBM-MSC treatment. Although additional studies are needed to clarify the exact therapeutic mechanisms of PEMF/hBM-MSC treatment, our findings provide a basis for the development of novel stroke rehabilitation strategies.